Analysis of Yeast RAD18 Gene Function. Interest in the mechanisms involved in maintaining the integrity of the eukaryotic genome has prompted an attempt to characterize the role of the RAD18 gene in the yeast Saccharomyces cerevisiae. Yeast strains with a mutant RAD18 gene exhibit an increased sensitivity to DNA damaging agents and enhanced spontaneous mutation rates, suggesting a role of the RAD18 gene product in DNA repair or replication or both. A recombinant plasmid containing RAD18 will be utilized to define the functional role of RAD18 gene in yeast DN metabolism. (a) The region of the recombinant plasmid coding for RAD18 gene product will be defined using transposon mutagenesis and/or subcloning to identify the minimal DNA region complementing the UV sensitivity of rad18-2 mutants, and this region of DNA will be sequenced. (b) Transposon insertion mutations or internal fragments subcloned into integrating vectors will be used to disrupt a RAD18 gene in diploids, which will then be sporulated and subjected to tetrad analysis to determine whether RAD18 is essential for survival. If essential, in vitro mutagenesis will be used to generate conditional lethal mutations to further define this gene's function. (c) The pattern of regulation of expression of RAD18 gene and gene product will be examined before and after DNA damaging treatments and during different phases of the cell division cycle by isolating mRNA from cells in these conditions and, using RAD18 promoter-lacZ fusion plasmids to measure beta- galactosidase levels form cells in these conditions. (d) RAD18 gene product will be overproduced following subcloning into and expression vector, and (e) the RAD18 gene product will be purified in order to perform biochemical assays for possible repair and/or replication functions.